Repeat proteins are found in all three phyla and present in some 6% of all eukaryotic protein coding sequences [1]. They are made up of tandem arrays of similar amino acid stretches that fold up into elongated architectures of repeating structural motifs that stack one upon the next producing extended superhelical structures [2]. Repeat proteins, by virtue of their inherent symmetries both in primary sequence and three dimensional structure, stand as remarkable models where the sequence-codes-folding-codes-function<hypothesis can be quantitatively evaluated. The energy landscape theory of protein folding is based on the principle of minimal frustration<[3]. This principle, however, does not rule out that some energetic frustration may be present in a folded protein. Moreover, the remaining frustration may facilitate motion of the protein around its native basin, and as such the residual frustration may be fundamental to protein function [4]. Recently developed methods for spatially localizing and quantifying the energetic frustration present in native protein structures reflects evolutionary constraints on a proteins<energy landscape and yields useful insights into their biological functions [5]. We focus our attention on the I:B proteins. These ankyrin-repeat (AR) containing proteins regulate the activity of the NF-:B transcription factor family, which is found misregulated in diseases such as cancer, arthritis, asthma, diabetes, AIDS and viral infections [6]. The Ankyrin-repeat region of I:B1 contains 6 ARs and displays a highly dynamic character when not complexed with NF-:B [7-9]. Moreover, it has been shown that its folding properties are clearly related to its function [10]. We envision a complementary approach to the following Aims presented in Project 3 of the parent grant: 1) characterize the overall kinetics of coupled folding and binding in the NF-:B/ I:B1 system. 2) characterize the residue specific dynamics of coupled folding and binding in the NF-:B/ I:B1 system. These require a joint theoretical and experimental effort that brings together computational analysis of the distributions of local frustration, its effect on the folding transitions (both simulated and measured), and how these affect the functional binding reactions. We will use bioinformatic methods to characterize the frustration patterns of various NF-:B and I:B family members [5], both in their free and bound states. We will use computational models to simulate the folding - binding reaction of I:B1 - NF-:B, using recently developed schemes that allow for energetic frustration [11]. We will design mutations that perturb the frustration distributions [11], realize those in the laboratory, and analyze how the structural transitions and the binding properties are affected by means of quantification of the appropriate thermodynamic observables [8,10,12]. This research will be done primarily in Buenos Aires, Argentina, at the Universidad Nacional de Quilmes, in collaboration with Dr Diego Ferreiro, as an extension of NIH Grant No P01-GM071862-01. PUBLIC HEALTH RELEVANCE: Transcription factors are proteins that control the synthesis of mRNA from genomes. Among transcription factors, the NF-:B transcription factor family, is found misregulated in diseases such as cancer, arthritis, asthma, diabetes, AIDS and viral infections [6]. This family is regulated by inhibitors called I:Bs and we have shown that I:B1;folds<upon binding to NF -:B [10]. The intricate folding transitions that this protein exhibits, the topic of the proposed research, may be a key to its biological functions, as they are likely to be related to its NF-:B recognition and inhibition rates [13], crucial processes in the overall behavior of the NF-:B signaling system [14].